How is tRNA processing organized in the cell nucleus? Available evidence suggests that tRNA processing is strictly ordered in vivo but not in vitro. By what mechanism(s) is tRNA processing order? We proposed to address this problem by focusing on the yeast tRNA ligase protein. To do this, we have first purified the ligase protein, isolated its gene, and demonstrated that it is essential in the cell. Ligase catalyzes part of the tRNA splicing reaction, which is the last observed nuclear processing event before transport of the tRNA from the nucleus to the cytoplasm. Ligase is localized to a site near the inner nuclear membrane, perhaps in a functional complex with other tRNA splicing enzymes. 1) We will use a combined physiological, genetic and biochemical approach to identify gene products with which ligase interacts in the cell, and to determine the domain of the ligase protein responsible for its interaction. 2) We will identify the remaining unidentified activity in the tRNA splicing reaction and determining whether or not ligase interacts with this gene product in a functional splicing complex. 3) Attempts will be made to reproduce ordered processing in isolated nuclei in order to examine in more detail the causes of ordering in the cell. These experiments represent an integrated approach at understanding critical events at the nuclear/cytoplasmic interface, the organization of tRNA processing in the nucleus, and its relation to tRNA transport.